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. 2022 Dec 23;24(1):174–185. doi: 10.1038/s41590-022-01366-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 3 — (a-f) Thymocytes and (g-j) LN cells from the indicated mice were analyzed by flow cytometry. (a-b) The percentage of cells at the DN3 stage (CD44⁻ CD25⁺) out of all DN (viable CD4⁻ CD8α^-) thymocytes is shown. (a) Representative mice. (b) Individual values and medians are shown. Lck^WT/WT: n = 22 mice in 10 independent experiments, Lck^KO/KO: 12/6, Lck^KR/KR: 12/6, Lck^CA/CA: 17/7, Lck^CA/KR: 11/5. (c) Percentages of mature SP4 (TCRβ⁺ CD24⁻CD4⁺ CD8α^-) and mature SP8 (TCRβ⁺ CD24⁻CD4⁻ CD8α⁺) thymocytes. Lck^WT/WT: n = 25 mice in 11 independent experiments, Lck^KO/KO: 13/7 independent experiments, Lck^KR/KR: 12/6 independent experiments, Lck^CA/CA: 18/8, Lck^CA/KR: 11/5, Lck^WT/KO: 16/7, Lck^WT/KR: 14/8, Lck^WT/CA: 19/8. Individual values and medians are shown. (d) Phosphorylation of TCRζ was in thymocytes from the indicated mice was analyzed by immunoblotting. A representative experiment out of 3 in total. (e-f) Phosphorylation of TCRζ in thymocyte subpopulations determined by flow cytometry. (e) A representative experiment out of 6 in total. (f) Normalized TCRζ phosphorylation (net gMFI of Lck^WT mice were set as 1). n = 6 independent experiments/mice. Means are shown. The statistical significance was determined using a Mann Whitney test. (g) Frequency of TCRβ⁺ LN cells was analyzed by flow cytometry. A representative experiment out of 11 for Lck^WT/WT, 7 for Lck^KO/KO, 6 for Lck^KR/KR, 8 for Lck^CA/CA and 5 for Lck^CA/KR mice in total. (h-i) A percentage of CD44⁺ CD49d⁻ T cells out of CD8⁺ T cells was determined by flow cytometry. (h) Representative samples of Lck^WT/WT and Lck^CA/CA mice are shown. (i) Individual values and medians are shown. Lck^WT/WT: 25 mice in 11 independent experiments, Lck^KO/KO: 13/7, Lck^KR/KR: 12/6, Lck^CA/CA:18/8, Lck^CA/KR: 10/5. (j) Frequency of regulatory T cells from overall CD4⁺ T cells in Lck variant mice is shown. n = 12 Lck^WT/WT, 14 Lck^CA/CA, 10 Lck^CA/KR mice in 3 independent experiments. Statistical significance was calculated using a Mann-Whitney test. (k) Ratio of CD4⁺/CD8⁺ T cell counts derived from bone marrows of Lck^WT/WT, Lck^CA/CA, and Lck^CA/KR donors in the mixed bone marrow chimeras (Fig. 1g-h). n = 13 Lck^WT/WT, 14 Lck^CA/CA, 13 Lck^CA/KR mice from 3 independent experiments. Individual values and medians are shown.

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